The traumatic spinal cord injury (SCI) is a public health problem associated with high mortality (Dryden D M, Saunders L D, Rowe B H, May L A, Yiannakoulias N, Svenson L W, Schopflocher D P, Voaklander D C. The epidemiology of traumatic spinal cord injury in Alberta, Canada. Can. J. Neurol. Sci. 2003; 30: 113-121) and severe consequences leading to disability and long and expensive rehabilitation treatments.
Besides having a high social and economic impact, it has been reported that, in the United States, Canada, Australia, Italy and Mexico the new annual cases of SCI are in a range of 18 to 55 per million people (Woodruff B A, Baron R C. A description of nonfatal spinal cord injury using a hospital-based registry. Am. J. Prev. Med. 1994; 10: 10-14, Dryden D M, Saunders L D, Rowe B H, May L A, Yiannakoulias N, Svenson L W, Schopflocher D P, Voaklander D C. The epidemiology of traumatic spinal cord injury in Alberta, Canada. Can. J. Neurol. Sci. 2003; 30: 113-121, O'-Connor P. Incidence and patterns of spinal cord injury in Australia. Accid. Anal. Prey. 2002; 34: 405-415, Pagliacci M C, Celani M G, Zampolini M, Spizzichino L, Franceschini M, Baratta S, Finali G, Gatta G, Perdon L; Gruppo Italiano Studio Epidemiologico Mielolesioni. An Italian survey of traumatic spinal cord injury. The Gruppo Italiano Studio Epidemiologico Mielolesioni study. Arch Phys Med Rehabil. 2003; 84: 1266-1275, Pardini M C. La epidemiología de la lesión medular traumática en el Distrito Federal. PhD thesis of the Secretaría de Salubridad y Asistencia 1998).
Victims suffering from SCI now have relief and rehabilitation programs designed to stop the long-term physical deterioration (Houle J D, Tessler A. Repair of chronic spinal cord injury. Exp Neurol. 2003; 182: 247-260). However, these individuals are waiting for a treatment that can restore their autonomic functions, decreasing neuropathic pain and recover their walking ability. Because of the importance of this disease, several institutions world wide have been formed who spend annually several hundred million dollars to support the research on this subject, however, until today there are no effective treatments (Houle J D, Tessler A. Repair of chronic spinal cord injury. Exp Neurol. 2003; 182: 247-260).
The pathophysiological events of the damage start with a process of self-destruction of the nervous tissue, abortive regeneration and healing around the site of injury (Aldskogius H, Kozlova E N. Strategies for repair of the deafferented spinal cord. Brain Res Brain Res Rev. 2002; 40: 301-308). During acute stage, an ischemic process is observed, due to damage to blood microcirculation that generates an energy failure which in turn translates into a loss of ionic regulation and edema caused by the mobilization of monovalent cations such as K+ and Na+ and divalent cations such as Ca2+, leading to spinal shock (Hulsebosch C E. Recent advances in patophysiology and treatment of spinal cord injury. Adv. Physiol. Educ. 2003; 26: 238-255).
The result of all these processes is a highly unfavorable environment for carrying out the healing processes of damaged tissue both in the injury site and in its periphery (Beattie M S, Farooqui A A, Bresnahan J C. Review of current evidence for apoptosis after spinal cord injury. J. Neurotrauma. 2000, 17: 915-925), the formation of cavities surrounded by an astroglial scar evolving to become multi-lobed cystic cavities functioning as a physical and chemical barrier that prevents axonal regeneration (Houle J D, Tessler A. Repair of chronic spinal cord injury. Exp Neurol. 2003; 182: 247-260; Profyris C, Cheema S S, Zang D, Azari M F, Boyle K, Petratos S. Degenerative and regenerative mechanisms governing spinal cord injury. Neurobiol Dis. 2004; 15: 415-436).
For years, tissue or cell transplants have been done after a SCI to promote axonal growth, the spinal cord regeneration and therefore the functional recovery (Zompa E A, Cain L D, Everhart A W, Moyer M P, Hulsebosch C E. Transplant therapy: recovery of function after spinal cord injury. J. Neurotrauma. 1997; 14: 479-506; Taoka Y, Okajima K. Spinal cord injury in the rat. Prog Neurobiol. 1998; 56: 341-358). Transplants using neural stem cells and multipotential precursor cells of embryo or fetus within the injury site have been performed (Stokes B T, Reier P J. Fetal grafts alter chronic behavioral outcome after contusion damage to the adult rat spinal cord. Exp Neurol. 1992, 116: 1-12, McDonald J W, Liu X Z, Qu Y, Liu S, Mickey S K, Turetsky D, Gottlieb D I, Choi D W. Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord. Nat Med 1999, 5: 1410-1412), transplant of fetal cells or fetal spinal cord tissue (Zompa E A, Cain L D, Everhart A W, Moyer M P, Hulsebosch C E. Transplant therapy: recovery of function after spinal cord injury. J. Neurotrauma. 1997, 14: 479-506, Coumans J V, Lin T T, Dai H N, MacArthur L, McAtee M, Nash C, Bregman B S. Axonal regeneration and functional recovery after complete spinal cord transection in rats by delayed treatment with transplants and neurotrophins. J. Neurosci. 200; 21: 9334-9344) and transplants of peripheral nerve and Schwann cells (Menei P, Montero-Menei C, Whittemore S R, Bunge R P, Bunge M B. Schwann cell genetically modified to secrete human BDNF promote enhanced axonal regrowth across transected adult rat spinal cord. Eur J Neurosci 1998, 10: 607-621). Transplants of oligodendrocytes to promote remyelinization (Jeffery N D, Crang A J, O'Leary M T, Hodge S J, Blakemore W F. Behavioral consequences of oligodendrocyte progenitor cell transplant into experimental demyelinating injury in the rat spinal cord. Eur J Neurosci 1999; 11: 1508-1514), the results have had a negative effect on regeneration because they express axonal growth inhibitory molecules (Tessier-Lavigne M, Goodman C S. Perspectives: neurobiology. Regeneration in the nogo zone. Science 2000 287: 813-814). Furthermore, transplants have been done using immature or non-reactive astrocytes to promote regeneration and increase remyelinization and reduce the formation of glial scar (Franklin R J, Crang A J, Blakemore W F. Transplanted type-1 astrocytes facilitate repair of demyelinating injuries by host oligodendrocytes in adult rat spinal cord. J Neurocytol 1991, 20: 420-430). Cells have been transplanted from microglia (Rabchevsky A G, Weinitzen J M, Coulpier M, Fages C, Tinel M, Junier M P. A role for transforming growth factor alpha as an inducer of astrogliosis. J. Neurosci. 1998, 18: 10541-10552), glial cells of the olfactory bulb to create a permissive environment for regeneration (Keyvan-Fouladi N, Li Y, Raisman G. How do transplanted olfactory ensheathing cells restore function? Brain Res Brain Res Rev. 2002; 40: 325-327) and even stem cells have been used, manipulated or not by genetic engineer to induce the synthesis of specific proteins such as neurotrophins, neurotransmitters, enzymes, extracellular matrix molecules and surface adhesion molecules, without obtaining good results in functional recovery (McDonald J W, Liu X Z, Qu Y, Liu S, Mickey S K, Turetsky D, Gottlieb D I, Choi D W. Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord. Nat Med 1999, 5: 1410-1412; Barami K, Diaz F G. Cellular transplant and spinal cord injury. Neurosurgery. 2000; 47: 691-700).
Despite these therapeutic attempts, most of transplants used in the treatment of SCI have failed, being unable to restore significantly the nerve function loss, leading to the development of new strategies as the use of biomaterials to try to repair the spinal cord. First reports worldwide show the use of carbon filaments implants acting as a bridge for the growth of damaged axons in the spinal cord of rats. Khan et al., (Khan T, Dauzvardis M, Sayers S. Carbon filament implants promote axonal growth across the transected rat spinal cord. Brain Res 1991; 541: 139-145) implanted a carbon filament in rats subjected to a complete section model of the spinal cord and they observed the axonal growth on and between the filaments, concluding that the carbon filaments could serve as a surface to bond favorably the growing axons as well as probably functioning as a mechanical guide.
The use of microspheres with nerve growth factor (NGF) encapsulated in ovalbumin joined to biodegradable polymers located at the site of injury, is a bioengineering technique developed in 1999 by Cao and Shoichet (Cao X, Schoichet M S. Delivering neuroactive molecularles from biodegradable microspheres for application in central nervous system disorders. Biomaterials. 1999, 20: 329-339), to promote axonal regeneration processes. This technology was tested in PC12 cells to determine bioactivity of NGF released. The results showed that NGF remains bioactive up to 91 days.
The biocompatible hydrogel of poly[N-(2-hydripropil) methacrylamide] (PHPMA), with a cell adhesion region of fibronectin Arg-Gly and Asp was synthesised and its rheological structure and dielectric properties were characterized by Woerly et al., (Woerly S, Pinet E, de Robertis L, Van Diep D, Bousmina M. Spinal cord repair with PHPMA hydrogel containing RGD peptides (NeuroGel). Biomaterials. 2001; 22: 1095-1111). This biomaterial was tested in a model of spinal cord injury by hemisection in Sprague-Dawley rats. The hydrogel was inserted within the spinal cord. The results showed that the hydrogel polymer provides a three-dimensional structure and continuity through the damaged area, facilitating the migration and reorganization of the cells. Angiogenesis and axonal growth were also observed within the microstructure and new tissue on it, as well as axonal growth going to the supraspinal area within the segment of spinal cord reconstructed, in addition, the presence of hydrogel decreased necrosis and cavities formation, reason why the authors point out that this polymer could be helpful in the injured spinal cord repair.
The use of polymers as tube-like structures that guide the growing axons and serve as a bridge between the transition zone, was a methodology used by Oudega et al., (Oudega M, Gautier S E, Chapon P, Fragoso M, Bates M L, Parel J M, Bunge M B. Axonal regeneration into Schwann cell grafts within resorbable poly(alpha-hydroxyacid) guidance channels in the adult rat spinal cord. Bio-materials. 2001, 22: 1125-1136). Reabsorbable polymers made of poly(D, L-lactic acid) (PLA50) with a co-polymer of high molecular weight of poly(L-lactic acid) mixed with 10% of oligomers of poly (L-lactic acid) (PLA100/10) were implanted in nervous tissue of Fisher strain adult rats that underwent a complete section of the spinal cord, in this study a 4-month follow-up was carried out. The results showed that since week 2, the tubes had spinal nervous tissue and blood vessels. Most of the myelinated axons were found 1 month after implantation. In this paper it is concluded that there is myelinated fibers growth within the implant made with PLA100/10 polymer, 2 months after being placed within the spinal cord. However, there was an evident decrease of this phenomenon 4 months later, reason why the authors recommend further studies to optimize this technique.
Another biodegradable material manufactured to facilitate the regeneration process and guide the growth of axons after a SCI, are the filaments made with poly-□-hydroxybutyrate (PHB) and fibronectin alginate+hydrogel. In a study conducted by Novikov et al., (Novikov L N, Novikova L N, Mosahebi A, Wiberg M, Terenghi G, Kellerth J O. A novel biodegradable implant for neuronal rescue and regeneration after spinal cord injury. Biomaterials. 2002; 23: 3369-3376), sectioning of the rubrospinal tract at the L1 vertebra level was performed, and it was observed that implantation of PHB reduces cellular death by 50%. The use of separate components has no effect on survival of neurons. Also, neonatal Schwann cells were added to transplant of PHB, observing regeneration of axons within the implant and throughout the nervous tissue, suggesting that the use of these biomaterials, plus Schwann cells, can serve as a neuronal support with an increased regeneration after SCI.
The use of hydrogel tubes made of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) (p(HEMA-co-MMA)), as potential axonal growth guidance channels in the central nervous system is another approach to restore spinal cord injury. The characteristics of these tubes are: softness and flexibility similar to a gel with interconnected macro-pores between the layers, controlled through a chemical formulation (Dalton P D, Flynn L, Shoichet M S. Manufacture of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) hydrogel tubes for use as nerve guidance channels. Biomaterials. 2002, 23: 3843-3851).
However, it has been shown that degradation of these polymers can cause an inflammatory reaction, although some of them are immunologically inert and especially resistant within the nervous system (Marchant R E, Anderson J M, Dillingham E O In vivo biocompatibility studies. VII. Inflammatory response to polyethylene and to a cytotoxic polyvinylchloride. J Biomed Mater Res 1986, 20: 37-50; Gautier S E, Oudega M, Fragoso M, Chapon P, Plant G W, Bunge M B, Parel J M. Poly(alpha-hydroxyacids) for application in the spinal cord: resorbability and biocompatibility with adult rat Schwann cells and spinal cord. J Biomed Mater Res 1998, 42: 642-654). Other authors have reported that for some polymers, adhesion of axons to these compounds is poor, however, Rangappa et al. (Rangappa N, Romero A, Nelson K D, Eberhart R C, Smith G M. Laminin-coated poly(L-lactide) filaments induce robust neurite growth while providing directional orientation. J Biomed Mater Res 2000, 51: 625-634), using nets coated with a matrix of laminin, were able to increase adhesion of axons to the matrix. Another strategy is the development of biomaterials with the capacity to bind encapsulated peptides (trophic factors, drugs, etc.), which are slowly released (Pechar M, Ulbrich K, Subr V, Seymour L W, Schacht E H. Poly(ethylene glycol) multiblock copolymer as a carrier of anti-cancer drug doxorubicin. Bioconjug Chem 2000, 11: 131-139) but not satisfactory results have been obtained. Collagen fibers alone or in combination with other materials have served as guide and support for axonal growth and for inducing regeneration (Heffner C D, Lumsden A G, O'Leary D D. Target control of collateral extension and directional axon growth in the mammalian brain. Science. 1990; 247: 217-220; Tong X J, Hirai K, Shimada H, Mizutani Y, Izumi T, Toda N, Yu P. Sciatic nerve regeneration navigated by laminin-fibronectin double coated biodegradable collagen grafts in rats. Brain Res 1994, 663: 155-162). The use of biocompatible materials to restore the damaged nervous tissue has advanced rapidly, developing materials that function as bridges to repair spinal cord, however, these polymers are synthesised traditionally by chemical methods or electrochemical polymerization (Wang J, Neoh K G, Kang E T. Comparative study of chemically synthesised and plasma polymerized pyrrole and thiophene thin films. Thin Solid Films 2004, 446: 205-217), which could interfere with their beneficial effects, since it has been shown that degradation of these polymers can cause an inflammatory reaction.
Biodegradable polymers synthesised from the mixture and combination of segments of pyrrole and thiophene with flexible aliphatic ester chains to facilitate their degradation, were described by Schmidt et al. (U.S. Pat. No. 6,696,575 B2, 2004), these ones have been proposed as an alternative treatment in the field of tissue engineering, given their chemical and electrical properties. These materials are flexible and its chemical structure allows free movement of electrons between the chains, increasing their conductive properties. In this patent, they are suggested as biomaterials to promote regeneration of peripheral nervous tissue of the spinal cord, as well as in other tissues (bone, muscle, etc.). However, evidences are not presented to support its use.
The use of plasma to obtain conductive polymer films is another methodology that has been used for the synthesis of polymers. During the synthesis process, monomers react in the gaseous phase of the procedure and do not need a chemical intermediary in the reaction. With this method, the chemical structure of polymers is different from that observed with chemical synthesis, showing higher purity, greater adherence and increased crossings and extensions (Wang J, Neoh K G, Kang E T. Comparative study of chemically synthesised and plasma polymerized pyrrole and thiophene thin films. Thin Solid Films 2004, 446: 205-217). Cruz et al (Cruz G J, Morales J, Olayo R. Films obtained by plasma polymerization of pyrrole. Thin Solid Films 1999, 342: 119-126) reported the plasma synthesis method of pyrrole-derived materials, among which those iodine-doped are found. These materials are not biodegradable, reason why their use in the nervous system would reduce inflammatory response conferring them higher efficiency, since several studies indicate that the inflammatory response is one of the mechanisms of secondary damage that destroy nerve tissue located in the periphery of the injury.
The conductive polymers are those materials formed by long chains of hydrocarbons with double bonds alternated or conjugated; which meet the electrical properties of metals and the mechanical properties of plastics. Its conductivity is mainly due to the addition of certain amounts of other chemicals products (doping), but also to the presence of conjugated double bonds that allow the passage of an electron flow. The doping technique involves the addition of atoms having electronegative properties. These atoms can act providing free electrons to polymeric bonds or subtracting electrons, which equals to generate positive charges. In both cases, the polymer chain becomes electrically unstable and if a potential difference is applied, electrons move through the polymer (Cruz G J, Morales J, Olayo R. Films Obtained by plasma polymerization of pyrrole. Thin solid films 1999, 342: 119-126). Although the physical mechanisms that change polymers toward conductors are not well known, the purity and organization of the polymer chains seem to have a great importance. Thus, when the structural organization of polymer is modified, the conductivity can be improved. The polymers used as conductors are composed mainly of carbon and hydrogen atoms, arranged in repeated monomer units, like any other polymer. In general, these units usually have a heteroatom as nitrogen or sulfur. The C atoms are linked together by an alternative set of single and double bonds ( . . . ═C—C═C—C═C— . . . ), i.e., they show hyperconjugation of bonds, this is a general characteristic of all conductive polymers. The conduction of electricity is due to the motion of electrons (e−). It is necessary for e− to move freely through the material. In solid conductors, e− move through discrete energy states called bands (originated from the extension of Molecular Orbital Theory to the entire solid-crystalline network). Solids can conduct electricity only if their last band is half full (good conductor or metallic conductor) or, if empty, it is found energetically near the last full band (semiconductor). If the jump filled-band→empty-band is energetically big this can be considered as an insulator (Cruz G J, Morales J, Olayo R. Films Obtained by plasma polymerization of pyrrole. Thin solid films 1999, 342: 119-126).
The polypyrrole (PPy) is a conductive polymer with a chemical structure positively charged to which various chemicals substances (dopants) can join to change its electrical properties. The PPy has been used as a biosensor (Lopez-Crapez E, Livache T, Marchand J, Grenier J. K-ras mutation detection by hybridization to a polypyrrole DNA chip. Clin Chem 2001; 47: 186-194), to detect glucose in blood, because of its biocompatibility. Furthermore, because of its conductive properties, it has been shown that PP has the capacity to stimulate proliferation of nerve cells (Kotwal A, Schmidt C E. Electrical stimulation alters protein adsorption and nerve cell interactions with electrically conducting biomaterials. Biomaterials. 2001; 22: 1055-1064), chromaffin cells (Aoki T, Tanino M, Sanui K, Ogata N, Kumakura K. Secretory function of adrenal chromaffin cells cultured on polypyrrole films. Biomaterials. 1996; 17: 1971-1974) and endothelial cells (Garner B, Georgevich A, Hodgson A J, Liu L, Wallace G G. Polypyrrole-heparin composites as stimulus-responsive substrates for endothelial cell growth. J Biomed Mater Res 1999, 44: 121-129). Schmidt et al., (Schmidt C E, Shastri V R, Vacanti J P, Langer R. Stimulation of neurite outgrowth using an electrically conducting polymer. Proc Natl Acad Sci U.S.A. 1997; 94: 8948-8953) showed that electrical stimulation increases neurite growth in PC-12 cells on PP films. Recent studies have demonstrated an acceptable biocompatibility of PP both in vitro (Zhang Z, Roy R, Dugre F J, Tessier D, Dao L H. In vitro biocompatibility study of electrically conductive polypyrrole-coated polyester fabrics. J Biomed Mater Res 2001, 57: 63-71) and in vivo (Jiang X, Marois Y, Traore A, Tessier D, Dao L H, Guidoin R, Zhang Z. Tissue reaction to polypyrrole-coated polyester fabrics: an in vivo study in rats. Tissue Eng. 2002; 8: 635-647), this background gives support for using the PPy and its derivatives in several biomedical and tissue engineering applications. Based on this information, it was decided to use two semiconductive polymers, the polypyrrole co-polymer with polyethylene glycol (PPy/PEG) and a polymer of iodine-doped polypyrrole (PPy/l) in a model of complete section of rat spinal cord.